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In this work, we find that CD8 + T cells expressing inhibitory killer cell immunoglobulin-like receptors (KIRs) are the human equivalent of Ly49 + CD8 + regulatory T cells in mice and are increased in the blood and inflamed tissues of patients with a variety of autoimmune diseases. Moreover, these CD8 + T cells efficiently eliminated pathogenic gliadin-specific CD4 + T cells from the leukocytes of celiac disease patients in vitro. We also find elevated levels of KIR + CD8 + T cells, but not CD4 + regulatory T cells, in COVID-19 patients, correlating with disease severity and vasculitis. Selective ablation of Ly49 + CD8 + T cells in virus-infected mice led to autoimmunity after infection. Our results indicate that in both species, these regulatory CD8 + T cells act specifically to suppress pathogenic T cells in autoimmune and infectious diseases.

In this work, we find that CD8

How the human immune system reacts prior to a cancer diagnosis is largely unknown. Here we analyze a longitudinal cohort of 133 healthy individuals (18 to over 90 years old), 28 of whom developed cancers over nine years. We find the global abundance of a broad spectrum of cytokines (26 out of 32 assayed) surges 2-3 years before cancer diagnosis but not with other conditions (such as inflammatory or cardiovascular diseases). These cytokine surges precede cancer diagnosis only in those 80 years or older, suggesting age-specific inflammatory effects. Meanwhile, in early-stage (but not late-stage) tissues across 10 cancer types, aging elevates cytokine transcriptional activity, which coincides with an age-dependent expansion of the inflammatory macrophage population. We further find that macrophage is a major producer of cytokines in cancer tissues, irrespective of cancer types. These results suggest that cancer associated macrophage is a major source of peripheral inflammatory molecules and may mobilize immunity well before a clinical diagnosis in the elderly. Citation Format: Guangbo Chen, Hong Zheng, Yael Rosenberg-Hasson, Saron Araya, Cindy Padilla, Kavita Sarin, Cornelia L. Dekker, Philip Grant, Holden T. Maecker, David Furman, Shai Shen-Orr, Purvesh Khatri, Mark M. Davis. Cytokines surge in the blood years prior to a cancer diagnosis in elderly individuals [abstract]. In: Proceedings of the AACR Special Conference: Aging and Cancer; 2022 Nov 17-20; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2022;83(2 Suppl_1):Abstract nr PR002.

While many diseases of aging have been linked to the immunological system, immune metrics capable of identifying the most at-risk individuals are lacking. From the blood immunome of 1,001 individuals aged 8–96 years, we developed a deep-learning method based on patterns of systemic age-related inflammation. The resulting inflammatory clock of aging (iAge) tracked with multimorbidity, immunosenescence, frailty and cardiovascular aging, and is also associated with exceptional longevity in centenarians. The strongest contributor to iAge was the chemokine CXCL9, which was involved in cardiac aging, adverse cardiac remodeling and poor vascular function. Furthermore, aging endothelial cells in human and mice show loss of function, cellular senescence and hallmark phenotypes of arterial stiffness, all of which are reversed by silencing CXCL9. In conclusion, we identify a key role of CXCL9 in age-related chronic inflammation and derive a metric for multimorbidity that can be utilized for the early detection of age-related clinical phenotypes. From the blood immunome of 1,001 individuals aged 8–96 years, the authors used deep learning to develop an inflammatory clock of aging (iAge) that tracks with multimorbidity, immunosenescence, frailty and cardiovascular aging, and is also associated with exceptional longevity in centenarians. The main contributor to iAge is the chemokine CXCL9, which is shown to control endothelial cell senescence and function.

Background Although intravenous immunoglobulin (IVIG) is effective therapy for Kawasaki disease (KD), the most common cause of acquired heart disease in children, 10–20% of patients are IVIG-resistant and require additional therapy. This group has an increased risk of coronary artery aneurysms (CAA) and there has been no adequately powered, randomized clinical trial in a multi-ethnic population to determine the optimal therapy for IVIG-resistant patients. Objectives The primary outcome is duration of fever in IVIG-resistant patients randomized to treatment with either infliximab or a second IVIG infusion. Secondary outcomes include comparison of inflammatory markers, duration of hospitalization, and coronary artery outcome. An exploratory aim records parent-reported outcomes including signs, symptoms and treatment experience. Methods The KIDCARE trial is a 30-site randomized Phase III comparative effectiveness trial in KD patients with fever ≥36 h after the completion of their first IVIG treatment. Eligible patients will be randomized to receive either a second dose of IVIG (2 g/kg) or infliximab (10 mg/kg). Subjects with persistent or recrudescent fever at 24 h following completion of the first study treatment will cross-over to the other treatment arm. Subjects will exit the study after their first outpatient visit (5–18 days following last study treatment). The parent-reported outcomes, collected daily during hospitalization and at home, will be compared by study arm. Conclusion This trial will contribute to the management of IVIG-resistant patients by establishing the relative efficacy of a second dose of IVIG compared to infliximab and will provide data regarding the patient/parent experience of these treatments.

Immune responses generally decline with age. However, the dynamics of this process at the individual level have not been characterized, hindering quantification of an individual’s immune age. Here, we use multiple ‘omics’ technologies to capture population- and individual-level changes in the human immune system of 135 healthy adult individuals of different ages sampled longitudinally over a nine-year period. We observed high inter-individual variability in the rates of change of cellular frequencies that was dictated by their baseline values, allowing identification of steady-state levels toward which a cell subset converged and the ordered convergence of multiple cell subsets toward an older adult homeostasis. These data form a highdimensional trajectory of immune aging (IMM-AGE) that describes a person’s immune status better than chronological age. We show that the IMM-AGE score predicted all-cause mortality beyond well-established risk factors in the Framingham Heart Study, establishing its potential use in clinics for identification of patients at risk. REPORTING SUMMARY. Further information on experimental design is available in the Nature Research Reporting Summary linked to this article.

Significance The immune system represents a compelling example of evolution in action: antibody diversity is created by a variety of molecular mechanisms, and then selection acts to preserve and propagate the most useful antibodies. We have combined immune repertoire sequencing with population genetics to measure the strength of selection on various antibody lineages in humans who have been vaccinated for influenza., Antibodies are created and refined by somatic evolution in B cell populations, which endows the human immune system with the ability to recognize and eliminate diverse pathogens. However, the evolutionary processes that sculpt antibody repertoires remain poorly understood. Here, using an unbiased repertoire-scale approach, we show that the population genetic signatures of evolution are evident in human B cell lineages and reveal how antibodies evolve somatically. We measured the dynamics and genetic diversity of B cell responses in five adults longitudinally before and after influenza vaccination using high-throughput antibody repertoire sequencing. We identified vaccine-responsive B cell lineages that carry signatures of selective sweeps driven by positive selection, and discovered that they often display evidence for selective sweeps favoring multiple subclones. We also found persistent B cell lineages that exhibit stable population dynamics and carry signatures of neutral drift. By exploiting the relationship between B cell fitness and antibody binding affinity, we demonstrate the potential for using phylogenetic approaches to identify antibodies with high binding affinity. This quantitative characterization reveals that antibody repertoires are shaped by an unexpectedly broad spectrum of evolutionary processes and shows how signatures of evolutionary history can be harnessed for antibody discovery and engineering.

Significance Chronic inflammation contributes to morbidity and mortality in aging, but whether similar mechanisms underlie dysfunction in infection-associated chronic inflammation is unclear. Using a multicohort systems immunology approach, we identified signatures of immune dysfunction that are shared in aging and chronic viral infections, namely HIV and hepatitis C virus. We show that these shared dysfunctions persist despite viral clearance, and we describe the changes in functional coordination that occur during viral eradication. Finally, we highlight a partial restoration in interferon-α sensitivity across all major immune cell lineages as viral load drops. Our findings suggest a broad and persistent functional remodeling and deterioration of the human immune system despite removal of a chronic pathogenic burden that shares features of chronic inflammation in aging., Chronic inflammation is thought to be a major cause of morbidity and mortality in aging, but whether similar mechanisms underlie dysfunction in infection-associated chronic inflammation is unclear. Here, we profiled the immune proteome, and cellular composition and signaling states in a cohort of aging individuals versus a set of HIV patients on long-term antiretroviral therapy therapy or hepatitis C virus (HCV) patients before and after sofosbuvir treatment. We found shared alterations in aging-associated and infection-associated chronic inflammation including T cell memory inflation, up-regulation of intracellular signaling pathways of inflammation, and diminished sensitivity to cytokines in lymphocytes and myeloid cells. In the HIV cohort, these dysregulations were evident despite viral suppression for over 10 y. Viral clearance in the HCV cohort partially restored cellular sensitivity to interferon-α, but many immune system alterations persisted for at least 1 y posttreatment. Our findings indicate that in the HIV and HCV cohorts, a broad remodeling and degradation of the immune system can persist for a year or more, even after the removal or drastic reduction of the pathogen load and that this shares some features of chronic inflammation in aging.

Beginning in December of 2019, a novel coronavirus, SARS-CoV-2, emerged in China and is now a global pandemic with extensive morbidity and mortality. With the emergence of this threat, an unprecedented effort to develop vaccines against this virus began. As vaccines are now being introduced globally, we face the prospect of millions of people being vaccinated with multiple types of vaccines many of which use new vaccine platforms. Since medical events happen without vaccines, it will be important to know at what rate events occur in the background so that when adverse events are identified one has a frame of reference with which to compare the rates of these events so as to make an initial assessment as to whether there is a potential safety concern or not. Background rates vary over time, by geography, by sex, socioeconomic status and by age group. Here we describe two key steps for post-introduction safety evaluation of COVID-19 vaccines: Defining a dynamic list of Adverse Events of Special Interest (AESI) and establishing background rates for these AESI. We use multiple examples to illustrate use of rates and caveats for their use. In addition we discuss tools available from the Brighton Collaboration that facilitate case evaluation and understanding of AESI.

A novel coronavirus (CoV), Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), emerged in late 2019 in Wuhan, China and has since spread as a global pandemic. Safe and effective vaccines are thus urgently needed to reduce the significant morbidity and mortality of Coronavirus Disease 2019 (COVID-19) disease and ease the major economic impact. There has been an unprecedented rapid response by vaccine developers with now over one hundred vaccine candidates in development and at least six having reached clinical trials. However, a major challenge during rapid development is to avoid safety issues both by thoughtful vaccine design and by thorough evaluation in a timely manner. A syndrome of “disease enhancement” has been reported in the past for a few viral vaccines where those immunized suffered increased severity or death when they later encountered the virus or were found to have an increased frequency of infection. Animal models allowed scientists to determine the underlying mechanism for the former in the case of Respiratory Syncytial virus (RSV) vaccine and have been utilized to design and screen new RSV vaccine candidates. Because some Middle East respiratory syndrome (MERS) and SARS-CoV-1 vaccines have shown evidence of disease enhancement in some animal models, this is a particular concern for SARS-CoV-2 vaccines. To address this challenge, the Coalition for Epidemic Preparedness Innovations (CEPI) and the Brighton Collaboration (BC) Safety Platform for Emergency vACcines (SPEAC) convened a scientific working meeting on March 12 and 13, 2020 of experts in the field of vaccine immunology and coronaviruses to consider what vaccine designs could reduce safety concerns and how animal models and immunological assessments in early clinical trials can help to assess the risk. This report summarizes the evidence presented and provides considerations for safety assessment of COVID-19 vaccine candidates in accelerated vaccine development.

Annual vaccination with influenza vaccines is recommended for protection against influenza in the United States. Past clinical studies and meta-analysis, however, have reported conflicting results on the benefits of annual vaccination. B-cell responses elicited following repeat influenza vaccinations over multiple seasons have not been examined in detail. We analyzed the B-cell and antibody (Ab) responses in volunteers vaccinated yearly, from 2010 or 2011 through 2014, with seasonal trivalent inactivated influenza vaccines. Statistical analyses were designed to help correct for possible bias due to reduced sample size in the later years of the study. We show that, after the second annual vaccination, the frequency of vaccine-specific plasmablasts and the binding reactivity of plasmablast-derived polyclonal Abs are reduced and do not increase in subsequent years. Similar trends are observed with the serum hemagglutination inhibition Ab response after each annual vaccination, as well as the binding reactivity of plasmablast-derived polyclonal Abs to the hemagglutinin of influenza A virus vaccine components, even with changes in the seasonal vaccine components during the study. Our findings indicate a diminished B-cell response to annual vaccination with seasonal trivalent influenza vaccine. These results emphasize the need for developing improved strategies to enhance the immunogenicity and efficacy of annual influenza vaccination.

Seasonal influenza vaccines elicit antibody responses that can prevent infection, but their efficacy is reduced in the elderly. While a subset of elderly individuals can still mount sufficient vaccine-induced antibody responses, little is known about the properties of the vaccine-induced antibody repertoires in elderly as compared to young responders. To gain insights into the effects of aging on influenza vaccine-induced antibody responses, we used flow cytometry and a cell-barcoding method to sequence antibody heavy and light chain gene pairs expressed by individual blood plasmablasts generated in response to influenza vaccination in elderly (aged 70 – 89) and young (aged 20 – 29) responders. We found similar blood plasmablast levels in the elderly and young responders seven days post vaccination. Informatics analysis revealed increased clonality, but similar heavy chain V(D)J gene usage in the elderly as compared to young vaccine responders. Although the elderly responders exhibited decreased antibody sequence diversity and fewer consequential mutations relative to young responders, recombinant antibodies from elderly responders bound a broader range of influenza strain HAs. Thus elderly influenza vaccine responders mount plasmablast responses with restricted diversity but with an increased breadth of binding across influenza strains. Our results suggest that the ability to generate plasmablast responses encoding cross-strain binding antibodies likely represents a mechanism important to vaccine responses in the elderly.

The elderly have reduced humoral immunity, as manifested by increased susceptibility to infections and impaired vaccine responses. To investigate the effects of aging on B-cell receptor (BCR) repertoire evolution during an immunological challenge, we used a phylogenetic distance metric to analyze Ig heavy-chain transcript sequences in both young and elderly individuals before and after influenza vaccination. We determined that BCR repertoires become increasingly specialized over a span of decades, but less plastic. In 50% of the elderly individuals, a large space in the repertoire was occupied by a small number of recall lineages that did not decline during vaccine response and contained hypermutated IgD+ B cells. Relative to their younger counterparts, older subjects demonstrated a contracted naive repertoire and diminished intralineage diversification, signifying a reduced substrate for mounting novel responses and decreased fine-tuning of BCR specificities by somatic hypermutation. Furthermore, a larger proportion of the repertoire exhibited premature stop codons in some elderly subjects, indicating that aging may negatively affect the ability of B cells to discriminate between functional and nonfunctional receptors. Finally, we observed a decreased incidence of radical mutations compared with conservative mutations in elderly subjects’ vaccine responses, which suggests that accumulating original antigenic sin may be limiting the accessible space for paratope evolution. Our findings shed light on the complex interplay of environmental and gerontological factors affecting immune senescence, and provide direct molecular characterization of the effects of senescence on the immune repertoire.

The T cell repertoire in each individual includes T cell receptors (TCRs) of enormous sequence diversity through the pairing of diverse TCR α- and β-chains, each generated by somatic recombination of paralogous gene segments. Whether the TCR repertoire contributes to susceptibility to infectious or autoimmune diseases in concert with disease-associated major histocompatibility complex (MHC) polymorphisms is unknown. Due to a lack in high-throughput technologies to sequence TCR α–β pairs, current studies on whether the TCR repertoire is shaped by host genetics have so far relied only on single-chain analysis. Using a high-throughput single T cell sequencing technology, we obtained the largest paired TCRαβ dataset so far, comprising 965,523 clonotypes from 15 healthy individuals including 6 monozygotic twin pairs. Public TCR α- and, to a lesser extent, TCR β-chain sequences were common in all individuals. In contrast, sharing of entirely identical TCRαβ amino acid sequences was very infrequent in unrelated individuals, but highly increased in twins, in particular in CD4 memory T cells. Based on nucleotide sequence identity, a subset of these shared clonotypes appeared to be the progeny of T cells that had been generated during fetal development and had persisted for more than 50 y. Additional shared TCRαβ in twins were encoded by different nucleotide sequences, implying that genetic determinants impose structural constraints on thymic selection that favor the selection of TCR α–β pairs with entire sequence identities.

Antibody memory protects humans from many diseases. Protective antibody memory responses require activation of transcriptional programs, cell proliferation, and production of antigen-specific antibodies, but how these aspects of the response are coordinated is poorly understood. We profiled the molecular and cellular features of the antibody response to influenza vaccination by integrating single-cell transcriptomics, longitudinal antibody repertoire sequencing, and antibody binding measurements. Single-cell transcriptional profiling revealed a program of memory B cell activation characterized by CD11c and T-bet expression associated with clonal expansion and differentiation toward effector function. Vaccination elicited an antibody clone which rapidly acquired broad high-affinity hemagglutinin binding during affinity maturation. Unexpectedly, many antibody clones elicited by vaccination do not bind vaccine, demonstrating non-specific activation of bystander antibodies by influenza vaccination. These results offer insight into how molecular recognition, transcriptional programs, and clonal proliferation are coordinated in the human B cell repertoire during memory recall.

Machine learning holds considerable promise for understanding complex biological processes such as vaccine responses. Capturing interindividual variability is essential to increase the statistical power necessary for building more accurate predictive models. However, available approaches have difficulty coping with incomplete datasets which is often the case when combining studies. Additionally, there are hundreds of algorithms available and no simple way to find the optimal one. Here, we developed Sequential Iterative Modelling “OverNight” or SIMON, an automated machine learning system that compares results from 128 different algorithms and is particularly suitable for datasets containing many missing values. We applied SIMON to data from five clinical studies of seasonal influenza vaccination. The results reveal previously unrecognized CD4+ and CD8+ T cell subsets strongly associated with a robust antibody response to influenza antigens. These results demonstrate that SIMON can greatly speed up the choice of analysis modalities. Hence, it is a highly useful approach for data-driven hypothesis generation from disparate clinical datasets. Our strategy could be used to gain biological insight from ever-expanding heterogeneous datasets that are publicly available.

BackgroundSeasonal influenza results in significant morbidity and mortality worldwide, but the currently licensed inactivated vaccines generally have low vaccine efficacies and could be improved. In this phase 1 clinical trial, we compared seasonal influenza vaccine regimens with different priming strategies, prime-boost intervals, and administration routes to determine the impact of these variables on the resulting antibody response.MethodsBetween August 17, 2012 and January 25, 2013, four sites enrolled healthy adults 18-70 years of age. Subjects were randomized to receive one of the following vaccination regimens: trivalent hemagglutinin (HA) DNA prime followed by trivalent inactivated influenza vaccine (IIV3) boost with a 3.5 month interval (DNA-IIV3), IIV3 prime followed by IIV3 boost with a 10 month interval (IIV3-IIV3), or concurrent DNA and IIV3 prime followed by IIV3 boost with a 10 month interval (DNA/IIV3-IIV3). Each regimen was additionally stratified by an IIV3 administration route of either intramuscular (IM) or intradermal (ID). DNA vaccines were administered by a needle-free jet injector (Biojector). Study objectives included evaluating the safety and tolerability of each regimen and measuring the antibody response by hemagglutination inhibition (HAI).ResultsThree hundred and sixteen subjects enrolled. Local reactogenicity was mild to moderate in severity, with higher frequencies recorded following DNA vaccine administered by Biojector compared to IIV3 by either route (p ConclusionsAll vaccination regimens were found to be safe and tolerable. While there were no overall differences between regimens, the DNA-IIV3 group by ID route, and the IIV3-IIV3 group by IM route, showed higher responses compared to the other same-route regimens.

Combining HLA-DQ-gluten tetramers with mass cytometry and RNA sequencing analysis, we find that gluten-specific CD4+ T cells in the blood and intestines of patients with celiac disease display a surprisingly rare phenotype. Cells with this phenotype are also elevated in patients with systemic sclerosis and systemic lupus erythematosus, suggesting a way to characterize CD4+ T cells specific for disease-driving antigens in multiple autoimmune conditions.

Machine learning has the potential to identify novel biological factors underlying successful antibody responses to influenza vaccines. The first attempts have revealed a high level of complexity in establishing influenza immunity, and many different cellular and molecular components are involved. Of note is that the previously identified correlates of protection fail to account for the majority of individual responses across different age groups and influenza seasons. Challenges remain from the small sample sizes in most studies and from often limited data sets, such as transcriptomic data. Here we report the creation of a unified database, FluPRINT, to enable large-scale studies exploring the cellular and molecular underpinnings of successful antibody responses to influenza vaccines. Over 3,000 parameters were considered, including serological responses to influenza strains, serum cytokines, cell phenotypes, and cytokine stimulations. FluPRINT, facilitates the application of machine learning algorithms for data mining. The data are publicly available and represent a resource to uncover new markers and mechanisms that are important for influenza vaccine immunogenicity., Measurement(s)immune response traitTechnology Type(s)digital curationFactor Type(s)gender • race • visit_age • bmi • flu_vaccination_history • statin_use • influenza_infection_history • influenza_hospitalization • cmv_status • ebv_statusSample Characteristic - OrganismHomo sapiens Machine-accessible metadata file describing the reported data: 10.6084/m9.figshare.9902447

The adaptive immune system's capability to protect the body requires a highly diverse lymphocyte antigen receptor repertoire. However, the influence of individual genetic and epigenetic differences on these repertoires is not typically measured. By leveraging the unique characteristics of B, CD4+ T and CD8+ T-lymphocyte subsets from monozygotic twins, we quantify the impact of heritable factors on both the V(D)J recombination process and on thymic selection. We show that the resulting biases in both V(D)J usage and N/P addition lengths, which are found in naïve and antigen experienced cells, contribute to significant variation in the CDR3 region. Moreover, we show that the relative usage of V and J gene segments is chromosomally biased, with ∼1.5 times as many rearrangements originating from a single chromosome. These data refine our understanding of the heritable mechanisms affecting the repertoire, and show that biases are evident on a chromosome-wide level., The diversity of antigen receptor specificities is largely generated by random recombination of its segments. Here the authors show, by genetic comparison of monozygotic twin lymphocyte subsets, that individual genetic and epigenetic biases also contribute to the shape of the B and T cell repertoires.

SUMMARY In an immune response, CD4+ T cells expand into effector T cells and then contract to survive as long-lived memory cells. To identify age-associated defects in memory cell formation, we profiled activated CD4+ T cells and found an increased induction of the ATPase CD39 with age. CD39+ CD4+ T cells resembled effector T cells with signs of metabolic stress and high susceptibility to undergo apoptosis. Pharmacological inhibition of ATPase activity dampened effector cell differentiation and improved survival, suggesting that CD39 activity influences T cell fate. Individuals carrying a low-expressing CD39 variant responded better to vaccination with an increase in vaccine-specific memory T cells. Increased inducibility of CD39 after activation may contribute to the impaired vaccine response with age., Graphical abstract

SUMMARY Antibody memory protects humans from many diseases. Protective antibody memory responses require activation of transcriptional programs, cell proliferation, and production of antigen-specific antibodies, but how these aspects of the response are coordinated is poorly understood. We profile the molecular and cellular features of the antibody response to influenza vaccination by integrating single-cell transcriptomics, longitudinal antibody repertoire sequencing, and antibody binding measurements. Single-cell transcriptional profiling reveals a program of memory B cell activation characterized by CD11c and T-bet expression associated with clonal expansion and differentiation toward effector function. Vaccination elicits an antibody clone, which rapidly acquired broad high-affinity hemagglutinin binding during affinity maturation. Unexpectedly, many antibody clones elicited by vaccination do not bind vaccine, demonstrating non-specific activation of bystander antibodies by influenza vaccination. These results offer insight into how molecular recognition, transcriptional programs, and clonal proliferation are coordinated in the human B cell repertoire during memory recall., Graphical Abstract, In Brief Antibody memory requires coordination of molecular recognition, gene expression programs, and clonal dynamics. Horns et al. study the human antibody memory response using single-cell and repertoire sequencing, revealing a transcriptional program of memory B cell activation, broadly binding anti-influenza antibodies, and widespread bystander activation of non-vaccine-binding antibodies after influenza vaccination.

Natural killer (NK) cells use a diverse array of activating and inhibitory surface receptors to detect threats and provide an early line of defense against viral infections and cancer. Here, we demonstrate that the cell surface protein CD38 is a key human NK cell functional receptor through a role in immune synapse formation. CD38 expression marks a mature subset of human NK cells with a high functional capacity. NK cells expressing high levels of CD38 display enhanced killing and IFN-γ secretion in response to influenza virus-infected and tumor cells. Inhibition of CD38 enzymatic activity does not influence NK cell function, but blockade of CD38 and its ligand CD31 abrogates killing and IFN-γ expression in response to influenza-infected cells. Blockade of CD38 on NK cells similarly inhibits killing of tumor cells. CD38 localizes and accumulates at the immune synapse between NK cells and their targets, and blocking CD38 severely abrogates the ability of NK cells to form conjugates and immune synapses with target cells. Thus, CD38 plays a critical role in NK cell immune synapse formation. These findings open new avenues in immunotherapeutic development for cancer and infection by revealing a critical role for CD38 in NK cell function.

Post-translational modifications of histone proteins and exchanges of histone variants of chromatin are central to the regulation of nearly all DNA-templated biological processes. However, the degree and variability of chromatin modifications in specific human immune cells remain largely unknown. Here, we employ a highly multiplexed mass cytometry analysis to profile the global levels of a broad array of chromatin modifications in primary human immune cells at the single-cell level. Our data reveal markedly different cell-type- and hematopoietic-lineage-specific chromatin modification patterns. Differential analysis between younger and older adults shows that aging is associated with increased heterogeneity between individuals and elevated cell-to-cell variability in chromatin modifications. Analysis of a twin cohort unveils heritability of chromatin modifications and demonstrates that aging-related chromatin alterations are predominantly driven by non-heritable influences. Together, we present a powerful platform for chromatin and immunology research. Our discoveries highlight the profound impacts of aging on chromatin modifications.

Background. Inactivated influenza vaccine (IIV) is recommended during pregnancy to prevent influenza infection and its complications in pregnant women and their infants. However, the extent to which pregnancy modifies the antibody response to vaccination remains unclear, and prior studies have focused primarily on hemagglutinin inhibition (HI) titers. A more comprehensive understanding of how pregnancy modifies the humoral immune response to influenza vaccination will aid in maximizing vaccine efficacy. Methods. Healthy pregnant women and control women were studied prior to, 7 days after, and 28 days after vaccination with IIV. HI titers, microneutralization (MN) titers, and the frequency of circulating plasmablasts were evaluated in pregnant versus control women. Results. Pregnant women and control women mount similarly robust serologic immune responses to IIV, with no significant differences for any influenza strain in postvaccination geometric mean HI or MN titers. HI and MN titers correlate, though MN titers demonstrate more robust changes pre- versus postvaccination. The induction of circulating plasmablasts is increased in pregnant women versus controls (median fold-change 2.60 vs 1.49 [interquartile range, 0.94–7.53 vs 0.63–2.67]; P = .03). Conclusions. Pregnant women do not have impaired humoral immune responses to IIV and may have increased circulating plasmablast production compared to control women.

Combining HLA-DQ-gluten tetramers with mass cytometry and RNA sequencing analysis, we find that gluten-specific CD4

Systemic sclerosis with pulmonary arterial hypertension (SSc-PAH) is a debilitating and frequently lethal disease of unknown cause lacking effective treatment options. Lymphocyte anomalies and autoantibodies observed in systemic sclerosis have suggested an autoimmune character. We study the clonal structure of the B cell repertoire in SSc-PAH using immunoglobulin heavy chain (IGH) sequencing before and after B cell depletion. We found SSc-PAH to be associated with anomalies in B cell development, namely, altered VDJ rearrangement frequencies (reduced IGHV2-5 segment usage) and an increased somatic mutation–fixation probability in expanded B cell lineages. SSc-PAH was also characterized by anomalies in B cell homeostasis, namely, an expanded immunoglobulin D–positive (IgD+) proportion with reduced mutation loads and an expanded proportion of highly antibody-secreting cells. Disease signatures pertaining to IGHV2-5 segment usage, IgD proportions, and mutation loads were temporarily reversed after B cell depletion. Analyzing the time course of B cell depletion, we find that the kinetics of naïve replenishment are predictable from baseline measurements alone, that release of plasma cells into the periphery can precede naïve replenishment, and that modes of B cell receptor diversity are highly elastic. Our findings reveal humoral immune signatures of SSc-PAH and uncover determinism in the effects of B cell depletion on the antibody repertoire.

The immune system consists of many specialized cell populations that communicate with each other to achieve systemic immune responses. Our analyses of various measured immune cell population frequencies in healthy humans and their responses to diverse stimuli show that human immune variation is continuous in nature, rather than characterized by discrete groups of similar individuals. We show that the same three key combinations of immune cell population frequencies can define an individual's immunotype and predict a diverse set of functional responses to cytokine stimulation. We find that, even though interindividual variations in specific cell population frequencies can be large, unrelated individuals of younger age have more homogeneous immunotypes than older individuals. Across age groups, cytomegalovirus seropositive individuals displayed immunotypes characteristic of older individuals. The conceptual framework for defining immunotypes suggested by our results could guide the development of better therapies that appropriately modulate collective immunotypes, rather than individual immune components.

Systemic sclerosis with pulmonary arterial hypertension (SSc-PAH) is a debilitating and frequently lethal disease of unknown cause lacking effective treatment options. Lymphocyte anomalies and autoantibodies observed in systemic sclerosis have suggested an autoimmune character. Here we study the clonal structure of the B-cell repertoire in SSc-PAH using immunoglobulin heavy-chain sequencing before and after B-cell depletion. We found SSc-PAH to be associated with anomalies in B-cell development, namely altered VDJ rearrangement frequencies (reduced IGHV2-5 segment usage) and an increased somatic mutation-fixation probability in expanded B-cell lineages. SSc-PAH was also characterized by anomalies in B-cell homeostasis, namely an expanded IgD+proportion with reduced mutation loads and an expanded proportion of highly antibody-secreting cells. Disease signatures pertaining to IGHV2-5 segment usage, IgD proportions and mutation loads were temporarily reversed after B-cell depletion. Analyzing the time course of B-cell depletion, we find that the kinetics of naïve replenishment are predictable from baseline measurements alone, that release of plasma cells into the periphery can precede naïve replenishment and that modes of B-cell receptor diversity are highly elastic. Our findings shed light on the humoral immune basis of SSc-PAH and provide insights into the effect of B-cell depletion on the antibody repertoire.AbbreviationsSSc-PAHSystemic sclerosis with pulmonary arterial hypertensionIGHimmunoglobulin heavy-chainBCRB-cell receptorCDR3complementarity-determining region 3IgimmunoglobulinAAamino acid

The 2009 pandemic due to influenza A(H1N1) virus (A[H1N1]pdm09) and recent human cases of infection due to highly pathogenic avian influenza A(H5N1) and A(H7N9) viruses emphasize the urgent need to adequately prepare for influenza pandemics. Vaccination is considered the most effective means to protect against influenza. Two types of seasonal influenza vaccines are currently available in the United States: inactivated influenza vaccine (IIV) and live, attenuated influenza vaccine (LAIV). In children 6 months to 18 years of age, LAIV has been consistently more efficacious than IIV against both antigenically matched and drifted strains [1–3]. LAIV was recently recommended as the preferred vaccine for healthy children 2–8 years of age by the Centers for Disease Control and Prevention's Advisory Committee on Immunization Practices [4]. In adults

Pregnant women experience increased morbidity and mortality after influenza infection, for reasons that are not understood. Although some data suggest that natural killer (NK)- and T-cell responses are suppressed during pregnancy, influenza-specific responses have not been previously evaluated. Thus, we analyzed the responses of women that were pregnant (n = 21) versus those that were not (n = 29) immediately before inactivated influenza vaccination (IIV), 7 d after vaccination, and 6 wk postpartum. Expression of CD107a (a marker of cytolysis) and production of IFN-γ and macrophage inflammatory protein (MIP) 1β were assessed by flow cytometry. Pregnant women had a significantly increased percentage of NK cells producing a MIP-1β response to pH1N1 virus compared with nonpregnant women pre-IIV [median, 6.66 vs. 0.90% (P = 0.0149)] and 7 d post-IIV [median, 11.23 vs. 2.81% (P = 0.004)], indicating a heightened chemokine response in pregnant women that was further enhanced by the vaccination. Pregnant women also exhibited significantly increased T-cell production of MIP-1β and polyfunctionality in NK and T cells to pH1N1 virus pre- and post-IIV. NK- and T-cell polyfunctionality was also enhanced in pregnant women in response to the H3N2 viral strain. In contrast, pregnant women had significantly reduced NK- and T-cell responses to phorbol 12-myristate 13-acetate and ionomycin. This type of stimulation led to the conclusion that NK- and T-cell responses during pregnancy are suppressed, but clearly this conclusion is not correct relative to the more biologically relevant assays described here. Robust cellular immune responses to influenza during pregnancy could drive pulmonary inflammation, explaining increased morbidity and mortality.

SummaryB cells produce a diverse antibody repertoire by undergoing gene rearrangements. Pathogen exposure induces the clonal expansion of B cells expressing antibodies that can bind the infectious agent. To assess human B cell responses to trivalent seasonal influenza and monovalent pandemic H1N1 vaccination, we sequenced gene rearrangements encoding the immunoglobulin heavy chain, a major determinant of epitope recognition. The magnitude of B cell clonal expansions correlates with an individual’s secreted antibody response to the vaccine, and the expanded clones are enriched with those expressing influenza-specific monoclonal antibodies. Additionally, B cell responses to pandemic influenza H1N1 vaccination and infection in different people show a prominent family of convergent antibody heavy chain gene rearrangements specific to influenza antigens. These results indicate that microbes can induce specific signatures of immunoglobulin gene rearrangements and that pathogen exposure can potentially be assessed from B cell repertoires.

Background. The immunological bases for the efficacies of the 2 currently licensed influenza vaccines, live attenuated influenza vaccine (LAIV) and inactivated influenza vaccine (IIV), are not fully understood. The goal of this study was to identify specific B-cell responses correlated with the known efficacies of these 2 vaccines. Methods. We compared the B-cell and antibody responses after immunization with 2010/2011 IIV or LAIV in young adults, focusing on peripheral plasmablasts 6–8 days after vaccination. Results. The quantities of vaccine-specific plasmablasts and plasmablast-derived polyclonal antibodies (PPAbs) in IIV recipients were significantly higher than those in LAIV recipients. No significant difference was detected in the avidity of vaccine-specific PPAbs between the 2 vaccine groups. Proportionally, LAIV induced a greater vaccine-specific immunoglobulin A plasmablast response, as well as a greater plasmablast response to the conserved influenza nuclear protein, than IIV. The cross-reactive plasmablast response to heterovariant strains, as indicated by the relative levels of cross-reactive plasmablasts and the cross-reactive PPAb binding reactivity, was also greater in the LAIV group. Conclusions. Distinct quantitative and qualitative patterns of plasmablast responses were induced by LAIV and IIV in young adults; a proportionally greater cross-reactive response was induced by LAIV.

Elderly humans show decreased humoral immunity to pathogens and vaccines, yet the effects of aging on B cells are not fully known. Chronic viral infection by cytomegalovirus (CMV) is implicated as a driver of clonal T cell proliferations in some aging humans, but whether CMV or Epstein-Barr virus (EBV) infection contributes to alterations in the B cell repertoire with age is unclear. We have used high-throughput DNA sequencing of immunoglobulin heavy chain (IGH) gene rearrangements to study the B cell receptor repertoires over two successive years in 27 individuals ranging in age from 20 to 89 years. Some features of the B cell repertoire remain stable with age, but elderly subjects show increased numbers of B cells with long CDR3 regions, a trend toward accumulation of more highly mutated IgM and IgG immunoglobulin genes, and persistent clonal B cell populations in the blood. Seropositivity for CMV or EBV infection alters B cell repertoires, regardless of the individual's age: EBV infection correlates with the presence of persistent clonal B cell expansions, while CMV infection correlates with the proportion of highly mutated antibody genes. These findings isolate effects of aging from those of chronic viral infection on B cell repertoires, and provide a baseline for understanding human B cell responses to vaccination or infectious stimuli.

Background Hypersensitivity disorders following vaccinations are a cause for concern. Objective To determine the type and rate by age, gender, and vaccine received for reported hypersensitivity reactions following monovalent 2009 pandemic influenza A (H1N1) vaccines. Design A systematic review of reports to the Vaccine Adverse Event Reporting System (VAERS) following monovalent 2009 pandemic influenza A (H1N1) vaccines. Setting/patients US Civilian reports following vaccine received from October 1, 2009 through May 31, 2010. Measurements Age, gender, vaccines received, diagnoses, clinical signs, and treatment were reviewed by nurses and physicians with expertise in vaccine adverse events. A panel of experts, including seven allergists reviewed complex illnesses and those with conflicting evidence for classification of the event. Results Of 1984 reports, 1286 were consistent with immediate hypersensitivity disorders and 698 were attributed to anxiety reactions, syncope, or other illnesses. The female-to-male ratio was ≥4:1 for persons 20-to-59 years of age, but approximately equal for children under 10. One hundred eleven reports met Brighton Collaboration criteria for anaphylaxis; only one-half received epinephrine for initial therapy. The overall rate of reported hypersensitivity reactions was 10.7 per million vaccine doses distributed, with a 2-fold higher rate for live vaccine. Limitations Underreporting, especially of mild events, would result in an underestimate of the true rate of immediate hypersensitivity reactions. Selective reporting of events in adult females could have resulted in higher rates than reported for males. Conclusions Adult females may be at higher risk of hypersensitivity reactions after influenza vaccination than men. Although the risk of hypersensitivity reactions following 2009 pandemic influenza A (H1N1) vaccines was low, all clinics administering vaccines should be familiar with treatment guidelines for these adverse events, including the use of intramuscular epinephrine early in the course of serious hypersensitivity reactions.

Malaria results in over 650,000 deaths each year; thus, there is an urgent need for an effective vaccine. Pre-clinical studies and recently reported human trials suggest that pre-erythrocytic stage vaccines can provide protection against infection. A Phase 1, randomized, placebo-controlled, dose-escalation study was conducted with a vaccine composed of a replication-deficient adenovirus-35 backbone with P. falciparum circumsporozoite (CS) surface antigen (Ad35.CS.01). Healthy adult subjects received three doses of 10 (8), 10 (9), 10 (10), or 10 (11) vp/mL Ad35.CS.01 vaccine or saline placebo intramuscularly at 0, 1, and 6-mo intervals. Adverse events were assessed and anti-CS antibody responses were determined by ELISA. Seventy-two individuals were enrolled, with age, gender, and ethnicity similar across each study arm. While the vaccine was generally well tolerated, adverse events were more frequent in the highest dose groups (10 (10) and 10 (11) vp/mL). More robust humoral responses were also noted at the highest doses, with 73% developing a positive ELISA response after the three dose series of 10 (11) vp/mL. The Ad35.CS.01 vaccine was most immunogenic at the highest dosages (10 (10) and 10 (11) vp/mL). Reactogenicity findings were more common after the 10 (11) vp/mL dose, although most were mild or moderate in nature and resolved without therapy.

Annual influenza vaccinations aim to protect against seasonal infections, and vaccine strain compositions are updated every year. This protection is based on antibodies that are produced by either newly activated or memory B cells recalled from previous encounters with influenza vaccination or infection. The extent to which the B-cell repertoire responds to vaccination and recalls antibodies has so far not been analyzed at a genetic level—which is to say, at the level of antibody sequences. Here, we developed a consensus read sequencing approach that incorporates unique barcode labels on each starting RNA molecule. These labels allow one to combine multiple sequencing reads covering the same RNA molecule to reduce the error rate to a desired level, and they also enable accurate quantification of RNA and isotype levels. We validated this approach and analyzed the differential response of the antibody repertoire to live-attenuated or trivalent-inactivated influenza vaccination. Additionally, we analyzed the antibody repertoire in response to repeated yearly vaccinations with trivalent-inactivated influenza vaccination. We found antibody sequences that were present in both years, providing a direct genetic measurement of B-cell recall.

Antibodies that bind to both H1 and H3 influenza strains exist in the pre-vaccination serum repertoire of healthy adults; most vaccine-elicited clonotypes bind either H1 or H3 strains. Molecular understanding of serological immunity to influenza has been confounded by the complexity of the polyclonal antibody response in humans. Here we used high-resolution proteomics analysis of immunoglobulin (referred to as Ig-seq) coupled with high-throughput sequencing of transcripts encoding B cell receptors (BCR-seq) to quantitatively determine the antibody repertoire at the individual clonotype level in the sera of young adults before and after vaccination with trivalent seasonal influenza vaccine. The serum repertoire comprised between 40 and 147 clonotypes that were specific to each of the three monovalent components of the trivalent influenza vaccine, with boosted pre-existing clonotypes accounting for ∼60% of the response. An unexpectedly high fraction of serum antibodies recognized both the H1 and H3 monovalent vaccines. Recombinant versions of these H1 + H3 cross-reactive antibodies showed broad binding to hemagglutinins (HAs) from previously circulating virus strains; several of these antibodies, which were prevalent in the serum of multiple donors, recognized the same conserved epitope in the HA head domain. Although the HA-head-specific H1 + H3 antibodies did not show neutralization activity in vitro, they protected mice against infection with the H1N1 and H3N2 virus strains when administered before or after challenge. Collectively, our data reveal unanticipated insights regarding the serological response to influenza vaccination and raise questions about the added benefits of using a quadrivalent vaccine instead of a trivalent vaccine.

Chronic inflammation, a decline in immune responsiveness, and reduced cardiovascular function areall associated with aging, but the relationships among these phenomena remain unclear. Here, we longitudinally profiled a total of 84 signaling conditions in 91 young and older adults and observed an age-related reduction in cytokine responsiveness within four immune cell lineages, most prominently Tcells. The phenotype can be partially explained by elevated baseline levels of phosphorylated STAT (pSTAT) proteins and a different response capacity of naive versus memory Tcell subsets to interleukin 6 (IL-6), interferon α (IFN-α), and, to a lesser extent, IL-21 and IFN-γ. Baseline pSTAT levels tracked with circulating levels of C-reactive protein (CRP), and we derived a cytokine response score that negatively correlates with measures of cardiovascular disease, specifically diastolic dysfunction and atherosclerotic burden, outperforming CRP. Thus, we identified an immunological link between inflammation, decreased cell responsiveness in the JAK-STAT pathway, and cardiovascular aging. Targeting chronic inflammation may ameliorate this deficiency in cellular responsiveness and improve cardiovascular function.